Dry chemistry, lateral flow-reconstituted chromatographic enzyme-driven assays

ABSTRACT

A lateral flow chromatographic assay format for the performance of rapid enzyme-driven assays is described. A combination of components necessary to elicit a specific enzyme reaction, which are either absent from the intended sample or insufficiently present therein to permit completion of the desired reaction, are predeposited as substrate in dry form together with ingredients necessary to produce a desired color upon occurrence of the desired reaction. The strip is equipped with a sample pad placed ahead of the substrate deposit in the flowstream, to which liquid sample is applied. The sample flows from the sample pad into the substrate zone where it immediately reconstitutes the dried ingredients while also intimately mixing with them and reacting with them at the fluid front. The fluid front moves rapidly into the final “read zone” wherein the color developed is read against predetermined color standards for the desired reaction. Pretreatment pads for the sample, as needed, (e.g. a lysing pad for lysing red blood cells in whole blood) are placed in front of the sample pad in the flow path as appropriate. The assay in the format of the invention is faster and easier to perform than analogous wet chemistry assays.  
     Specific assays for glucose-6-phosphate dehydrogenase (“G-6PD”), total serum cholesterol, β-lactamase activity and peroxidase activity are disclosed.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/370,574 filed Feb. 24, 2003.

[0002] The present invention relates to conducting rapid, dry-chemistry,enzyme-driven chemistry assays using lateral flow chromatography.

BACKGROUND OF THIS INVENTION

[0003] The human enzyme glucose-6-phosphate dehydrogenase (“G6PD”)performs a critical function in human biochemistry. It is part of theoxidative pentose pathway, wherein it functions to minimize oxidativeattacks of free radicals upon cells by providing reducingequivalents-i.e., G6PD converts glucose-6-phosphate to6-phosphoglutonate, thereby liberating a proton that reducesnicotinamide adenine dinucleotide phosphate, NAPD, to NAPDH. The NAPDHinitiates a series of downstream reactions that ultimately reduce thefree radical oxidizing agents and render many of them ineffective innormal human biochemistry.

[0004] G6PD is present in all human cells, but it is in higherconcentration in red blood cells which, in one of their primaryfunctions, act as oxygen transport vehicles and are hence particularlysusceptible to oxidative attack. The efficiency of the G6PD system isremarkably high as reflected by the fact that during normal activityless than 1% of its capacity is utilized in combating and preventingundesirable oxidative effects. However, when strong oxidizing agents,such as members of the quinine class of anti-malarial drugs must beintroduced to humans, the need for rapid production of reducing agentsis greatly increased.

[0005] Several mutations of the gene which encodes for G6PD are knownwhich decrease the efficiency of the enzymes in the biochemistry ofindividuals processing such a mutation in both halves of their genome,causing the quantity of their G6PD to remain at the same level as inpeople with a normal gene, but also causing their G6PD to show greatlyreduced specific activity. In these individuals, administration ofstrong oxidizing agents such as members of the class of quinine-typeanti-malarials, may cause severe clinical complications, such ashemolytic anemia, because the low specific activity of their G6DP doesnot enable the production of sufficient reducing agents to prevent rapidunwanted oxidative effects on their red blood cells. In areas wheremalarial infections are common and at times even epidemic, a needtherefore exists for a rapid efficient test that will readilydistinguish persons having G6PD of low specific activity from personswhose G6PD activity is normal and will enable medical personnel toensure that (1) the quinine antimalarials are prescribed only forindividuals with normal or better G6PD specific activity and (2) personswith lower than normal G6PD activity are medicated with an alternativetype of anti-malarial drugs.

[0006] Heretofore, assays that involve enzyme activity, in any context,have most usually been conducted in “wet chemistry” formats whichrequire trained laboratory personnel to prepare for and perform them.The reagents for such assays must either be made fresh from drycomponents or be reconstituted from commercially available driedformulations. Wet reagents are less stable than dried ingredients ordried formulations and, to the extent they must be stored, morestringent, carefully monitored storage conditions, including specialhandling techniques to prevent contamination, are required. Those assaysalso require instruments such as spectrophotometers, fluorimeters orother such instrumental equipment to read the endpoint results of theassay. Such assays are not practical for use in doctor's offices,hospitals and nursing home facilities, under epidemic conditions, or forhome or field use.

[0007] Automated clinical chemistry analyzer systems are in industrialuse which perform dry chemistry formatted assays wherein the presence,absence, concentration or specific activity of a substance present in orabsent from a sample is determined. Such a substance is, for purpose ofthis application, referred to as the “analyte” and it may be an enzymeper se (as in the G6PD assay hereinafter described in detail) or asubstance necessary to the elicitation of a specific enzyme activity.Examples of automated clinical chemistry analyzer systems are theJohnson and Johnson Vitros™ and the Roche Cobas™ systems. These andsimilar automated systems are not subject, when performing as designed,to the preparation skill requirements and shelf life problems associatedwith humanly performed dry chemistry assay work. Because programmedrobots perform the manipulative tasks, the need for intensively trainedhumans is likewise avoided. The systems, however, require on boardreader instrumentation and they are necessarily too large, toocomplicated and generally too burdened with infrastructure requirementsto be practical for use in doctor's offices or homes and in manyhospitals, clinics and like places. Clearly, they have too manytechnical requirements for field use.

[0008] There are available, as well, a very few non-instrument baseddry-chemistry assays, such as the Orasure QED™ assay for alcohol whichis based upon use of the alcohol dehydrogenase enzyme to determinealcohol content of saliva in the field. This and other known assays ofthis genre have heretofore been limited to determinations that can bemade on samples that are free of substances that may obscure, inhibit orin some other manner intrinsically interfere with and render imprecisedeterminations that are dependent upon some aspect of enzymatic actionor content.

[0009] An example of an enzymatic assay that operates on samplescontaining visually obscuring substances and uses antibody capture zonesto select for enzyme analytes is shown in U.S. Pat. No. 5,506,114. Thissystem requires wash steps to remove the visually obscuring substancesand is sufficiently cumbersome to perform that it is impractical forfield use or use in doctor's offices, homes, most clinics and manyhospitals and the like.

BRIEF DESCRIPTION OF THE INVENTION

[0010] In its broadest aspect, this invention rests upon the recognitionthat rapid, dry chemistry, enzyme-driven assays may advantageously beconducted using lateral flow chromatography, wherein predeposited drysubstrate, as hereinafter defined, is reconstituted chromatographicallyby the lateral flow of liquid sample and entrained substrate through atleast one region of a lateral flow device, with production of acolorimetric reaction at the forward flow front of the sample-substratemixture in the endpoint or “read” zone of the chromatographic device.The color produced is that typical of the endpoint color of thecorresponding wet chemistry clinical assay, obtained in the region ofthe device where forward flow ceases, i.e., the zone that is farthestfrom the point of sample introduction. It is within the scope of theinvention, depending upon the specific assay being conducted, to includechromatographic regions in the device that remove interfering substancespresent in the sample and/or regions that have been treated topreconcentrate the analyte before it moves into the endpoint reactionzone. In some assays, at least one substance heretofore deemed tointerfere with the endpoint observation, i.e., hemoglobin, need not beremoved since its otherwise endpoint-obscuring color deposits equally inthe endpoint zone and the zone just preceding it. The result in thiscase is that the endpoint is easily observable by direct comparison ofthe color produced in the endpoint zone with that of the unreacted redcolor in the abutting, immediately preceding zone.

[0011] In general, in the lateral chromatography, enzyme-driven assaysof this invention, the movable, predeposited dry subtrate is placed nearto and just beyond the junction of the sample receiving pad and the nextpad in the sample flow path on the chromatographic strip. It may,however be placed elsewhere in the sample flow path to accommodateparticular requirements of either the sample or one or more ingredientsin the subtrate, so long as it is placed in the flow path substantiallybefore the endpoint, or “read”, zone where sample flow stops and anyexcess fluid present runs off into an absorption pad or other sinkdevice that may be provided.

[0012] It is important that the dried substrate be deposited within atightly confined area so as to facilitate its being completely picked upby the forward flow of the sample. The placement in the flow path of thedried subtrate should also take into consideration that reconstitutionof the subtrate in dissolved or dispersed form within the liquid sampleis desirably completed by the time the sample reaches the point wheresample flow ceases.

[0013] It has also been found that the present enzyme-driven lateralflow assay can be combined with known solid phase isolation methods, andthat in at least some instances when this is done, the sensitivity ofdetection of the desired end point is enhanced substantially.

[0014] The format of such methodology involves binding a ligand for thetarget enzyme to a particulate solid support material—e.g., disks offilter paper or other common solid support material, such as but notlimited to nitrocellulose, nylon, polyethylene, etc. orsuperparamagnetic particles and the like—by coupling, coating,impregnation or any other known method. The ligand-bound particles arethen mixed with a sample of fluid known to contain the target enzyme andincubated for a period requisite to allow binding of target enzyme tothe ligand. The particles containing enzyme-ligand reaction productsbound thereto are then separated from the fluid sample by knownseparation techniques, including filtration, sedimentation,centrifugation and in the case of superparamagnetic particles,subjection to the influence of a gradient magnetic field of sufficientstrength.

[0015] The collected particles containing bound enzyme-ligand reactionproduct, after separation from the initial sample, are then suspended ina volume of known buffer selected as one known to be appropriate to theenzyme-ligand reaction product and this buffer suspension is utilized asthe sample in an enzyme-driven test assay constructed for determinationof enzyme concentration or some other parameter of the enzyme. Thisapproach is of special value in a method for beta-lactamase enrichmentand detection, wherein all beta-lactamase present in a bacterial cultureor in sample of human fluid, such as a nasal wash, or a urine sample, ispreconcentrated by immunological separation via ligand-bound particlesand is then transferred into the uptake volume of the enzyme-driventest.

[0016] For convenience of shipping, storage and use, eachchromatographic strip of this invention is preferably housed within asuitable device constructed so that the strip is positioned laterally.Many such devices are well-known in the art and any of them constructedso that the performance of an assay on the chromatographic strippositioned within it is performed by lateral flow may appropriately beutilized.

[0017] This format for conducting enzyme-driven assays has a number ofadvantages as hereinafter described in detail.

[0018] A specific G6PD assay that can easily be used successfully byanyone operating in the field, the home, a doctor's office or at anysite where trained laboratory personnel and instrumentation are lacking,is specifically described hereinafter and depicted in the accompanyingdrawings, as are assays for total serum cholesterol, beta-lactamaseactivity, and peroxidase activity.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0019]FIG. 1A represents a chromatographic strip preprepared for theperformance of the G6PD assay of this invention.

[0020]FIG. 1B shows the same strip after the sample has been applied toit and before the sample has reached the endpoint zone.

[0021]FIG. 1C shows the strip as it appears when the assay is completed.

[0022]FIG. 2A is a chart showing data obtained from use of the G6PD testof this invention.

[0023]FIG. 2B is a graph of time in seconds elapsed from the cessationof forward flow at the end of the strip to the appearance of purplishblue color in the endpoint zone, plotted against G6PD activity level forthe samples, measured activity level of which is shown in the FIG. 2Achart.

[0024]FIG. 3A is a chart showing data obtained in the test of thisinvention for total serum cholesterol.

[0025]FIG. 3B A plot of time in seconds elapsed from cessation offorward flow at the end of the strip to appearance of blue/purple colorin the “read” or endpoint zone, versus total serum cholesterol inmg./deciliter.

[0026]FIG. 4A is a chart showing data obtained with whole blood in thetotal serum cholesterol test, as described in Example 2 hereof.

[0027]FIG. 4B is a plot of time in seconds to appearance of bluishpurple color against cholesterol in mg. per dl.

[0028]FIG. 5A is a chart of data obtained using strips of this inventionin measuring β-lactamase activity, using commercially availableβ-lactamase standards.

[0029]FIG. 5B is a plot of time in seconds to appearance of bluishpurple color against standard activity in U/ml, where “U” representsunits of activitiy.

[0030]FIG. 6A is a chart obtained from measuring β-lactamase activity ina culture of E. coli known to produce β-lactamase.

[0031]FIG. 6B is a graph of the data from FIG. 6A showing time inseconds to appearance of bluish purple color against β-lactamaseactivity measured in CFU (colony-forming units) per ml.

[0032]FIG. 7 is a chart of the data from the measurement of peroxidaseas described in Example 4, infra.

DETAILED DESCRIPTION OF THE INVENTION

[0033] For purposes of describing this invention in its broadest scope,the following four definitions apply wherever the terms appear in thisapplication:

[0034] (1). “Sample” refers to any liquid biological or environmentalmatrix or any liquid extract or liquid concentrate thereof that is to beassayed.

[0035] (2). “Analyte” refers to a target substance of the assay whichmay be present in, or absent from, the sample. The analyte may itself bean enzyme or it may be a substance required to elicit specific enzymeactivities, such as substrate, co-substrate or cofactor.

[0036] (3). “Substrate” refers to a combination of those componentsnecessary to elicit a specific enzyme reaction, which components are notpresent in the sample or are present therein in insufficient quantitywhere the analyte is not an enzyme, or where the initial enzyme reactiondrives a cascade of additional reactions requisite to the desired finaldetermination. The substrate may contain any combination of cofactors,substrates as defined in the preceding sentence, co-substrates, dye orcolorimetric components, or enzymes themselves.

[0037] (4). The term “dry-chemistry” refers to an assay format where thecomponents required for a given determination on a sample are maintainedin dry form until reconstituted by the performance of the assay itself,rather than being reconstituted prior to and separate from the assayprocedures.

[0038] Reconstituting substrate components necessary in enzyme-drivenassays by lateral flow chromatography exhibits a host of advantages incomparison to methods that do not utilize such chromatography. At leastsome of them are identified below:

[0039] In the usual manual performance of enzyme driven assays, it isnecessary to dilute the sample serially in order to control rapid enzymekinetics; in the lateral flow assays of this invention, sample dilutionis unnecessary because only a very small sample is applied to thechromatographic strip upon which the assay is performed.

[0040] The sample itself reconstitutes the substrate, therebyeliminating the need for separate reconstitution buffers and steps.

[0041] This chromatographic reconstitution of the subtrate increases thesubstrate concentration available to the sample over that provided inthe usual laboratory performance of a comparable assay.

[0042] The chromatographic media utilized are any of those well-knownand well-characterized in the art. Their known characteristics mayreadily be taken advantage of, in particular assays where this isdesirable, by reserving a zone of the strip in which to concentrateanalyte by removing some of the fluid present therein or by reserving azone of the strip and pretreating it appropriately so that substancespresent in the sample that tend to inhibit or interfere with the desiredenzyme reaction are wholly or partially immobilized in that zone, ortheir flowability is retarded there.

[0043] Also, inasmuch as the endpoint color formation is restricted to asingle endpoint zone, it is much easier to read and evaluate, evenwithout instruments such a spectrophotometers, than when color isdiffused throughout a large liquid volume.

[0044] Further, the action of the sample in picking up dried substrateat its fluid front results in leaving an essentially substrate free zoneimmediately adjacent to the endpoint zone. As the sample moves forward,there is clear differentiation between any residual color fromhemoglobin and the desired endpoint color, forming at the fluid front.This eliminates any need, in some cases, to remove any substance in thesample (such as the red of hemoglobin) that is known to produce visuallyobscuring color when manual chemistry tests with liquid samples areperformed.

[0045] Still further, the relative speed from sample introduction toendpoint reaction that characterizes chromatographic lateral flow testsprovides great advantages in the enzyme driven tests. These tests, whenconducted by classical manual analytical methods often require digestiontimes for contact between sample and substrate that are in the order of30-45 minutes and even longer. Notably, these times do not include thetime needed in these classical manual methods for making dilutions,conducting concentration steps or substance removal steps, and the likemanipulations. By contrast, the lateral flow assays can usually beconducted in time periods within a 5-20 minute range, starting from theintroduction of the sample to the strip and ending with evaluation ofthe endpoint result.

[0046] To measure an analyte in red blood cells, such as the G6PDanalyte of Example 1 herein, the red blood cells must be lysed (splitopen) with surfactants or other lysing agents before the analyte can bemeasured. Another such analyte normally found in red blood cells, anassay for measuring which in the dry chemistry format of this inventioncan readily be devised, is pyruvate kinase.

[0047] There are also many instances where the analyte is normallypresent in blood serum or plasma, rather than red blood cells, that areenzyme-driven and may beneficially be converted to the “dry chemistry”format of this invention. Among them are such tests as those forglucose, cholesterol, HDL-cholesterol, triglycerides, urea nitrogen,creatinine, alanine aminotransferase (ALT), aspartate aminotransferase(AST), lactate dehydrogenase (LDH), creatinine kinase (CK) and the like.

[0048] The present invention will likewise be useful in instances wherethe analyte is present in other biological fluids, such as measuringalcohol in saliva or measuring drugs such as acetaminophen or salicylatein urine.

[0049] Overall, the present invention is perceived as having specialutility in situations where:

[0050] (A). the typical concentration of analyte in the sample is verylow such as in the creatinine and uric acid test for measuring kidneyfunction; where the intimate mixture of sample and substrate at thefluid front will improve the sensitivity of the test compared to that ofclassical manual methods;

[0051] (B). screen testing providing a qualitative “yes” or “no” resultis conducted to determine the onset or severity of a disease, such asthe ALT (alanine aminotransferase) and AST (aspartate aminotransferase)screens for liver damage, especially on patients who are taking drugscapable of causing liver damage, and screens conducted on infants forgenetically caused disorders, such as the test for phenylketonuria (PKU)or the test for galactose used to detect galactosemia; and the like; and

[0052] (C). tests where some portion of a sample needs to be removedprior to measurement of another portion-e.g. the measurement of HDL(highdensity lipids)-cholesterol, wherein LDL (low density lipids) and VLDL(very low density lipids) need to be removed, by precipitation orotherwise, before total HDL-cholesterol is measured.

[0053] As is apparent from the foregoing, the invention is adaptable toboth qualitative and quantitative formats. Some other milieus, inaddition to those mentioned above, in which it is amenable to beingwidely applied are those wherein enzyme-labeled antibodies have beenused in wet chemistry methods, to detect the presence of a suspectedantigen in an unknown sample, followed by reacting enzyme-taggedantibody-antigen reaction product with an appropriate color-producingagent. These tests have been traditionally performed both qualitativelyand quantitatively in wet chemistry formats. Adapting them to beingperformed according to this invention will produce all the benefits ofspeed, ease of manipulation, and the like that are noted hereinforeother enzyme-driven reactions.

[0054] Particular attention has been given to date to the application ofthis invention to whole blood chemistry tests.

[0055] For example, glucose may be determined by a reductive assay oftenutilized in blood taken from diabetic patients. To perform this assayaccording to the present invention, the immunochromatographic strip isprepared with predeposited dry ingredients consisting of glucosedehydrogenase, nicotinamide adenine dinucleotide (“NAD”), nitro bluetetrazolium and diaphorase. Glucose reduces to its hydride, and thehydride in the presence of diaphorase reduces nitro blue tetrazolium toprovide purplish blue color in the “read” zone.

[0056] In the oxidative assay for glucose the dry ingredients depositedin the substrate zone are glucose oxidase, peroxidase, 4-aminoantipyrineand phenol, or a derivative of phenol. Glucose and glucose oxidase inthe presence of oxygen produce hydrogen peroxide, which in turn reactswith aminopyrine and phenol (or a phenol derivative) to produce adistinct color in the “read” zone.

[0057] A cholesterol assay often utilized in performing a blood lipidprofile is conducted according to this invention by depositingcholesterol esterase, cholesterol oxidase, 4-amino-antipyrine and aphenol derivative, all in dry form, in the substrate zone. When a bloodsample is introduced to the strip, lysed and allowed to flow along thestrip, a distinct color is observed in the read zone that has anintensity proportional to the cholesterol concentration in the sample.This permits the development of color standards by well known methods.Such standards are conveniently used in situations where instruments forreading color intensity are not readily available, e.g doctor's offices,the home, in the field, etc.

[0058] A test according to this invention for measuring HDL-cholesterolemploys a preprepared ICT strip wherein a first zone immediatelyfollowing the “lyse” zone is provided with deposited, immobilizedprecipitating reagents that capture and bind the low density (“LDL”) andvery low density lipids (“VLDL”) in the sample, allowing only theHDL-cholesterol in the sample to pass into the substrate zone. Theingredients in the substrate zone are again dry cholesterol esterase,cholesterol oxidase, 4-aminoantipyrine and a phenol derivative and thecolor produced in the “read” zone is proportional in intensity to theconcentration of HDL-cholesterol in the sample.

[0059] To perform the creatinine assay for kidney malfunction assessmentby the method of this invention, the predeposited dry ingredients in thesubstrate zone are creatinine immunohydrolase, sarcosine oxidase,peroxidase, 4-aminoantipyrine and a phenol derivative.

[0060] An ALT assay for liver malfunction can be performed according tothis invention on an ICT strip wherein the dry ingredients predepositedin the substrate zone are pyruvate oxidase, peroxidase,4-aminoantipyrine and a phenol derivative.

[0061] Preparing an ICT strip as herein described with predeposited dryingredients in the substrate zone consisting of creatinine, adenosinetriphosphate, phosphophenol pyruvate, pyruvate oxidase, peroxidase,4-aminoantipyrine and a phenol derivative enables performance accordingto this invention of an assay for congestive heart failure.

[0062] Where phenylketonuria (PKU) is suspected in neonates, thisinvention provides a convenient “yes-no” assay wherein an ICT strip isequipped with a dry deposit of phenylalanine dehydrogenase, nicotinamideadenine dinucleotide and diaphorase. When a blood sample is added andcolor appears in the read zone, the test is positive for PKU; if nocolor appears, the disease is not present.

[0063] Tests for alcohol content in blood can be run both oxidativelyand reductively in the method of this invention. A test strip for thereductive test can be prepared by depositing alcohol dehydrogenase,nicotinamide adenine dinucleotide, nitro bluetetrazolium and diaphorasein dry form in the substrate zone; an oxidative test for the samepurpose employs an ICT strip in the substrate zone of which ispredeposited dry alcohol oxidase, peroxidase, 4-aminoantipyrine and aphenol derivative. In either case, the color in the endpoint, or “read”zone will be proportional to the alcohol content of the blood sample.

[0064] An assay for acetaminophen (Tylenol R) in blood can convenientlybe run according to this invention. The ICT strip for this purpose willcontain predeposited dry aryacylaminidase, ortho-cresol, and anoxidizing agent, such as sodium bisulfite. When a blood sample is addedone can determine whether or not the patient has overdosed on theacetaminophen by comparing its color intensity to that of apreestablished color standard.

[0065] A rapid dry-chemistry lateral flow assay was devised toillustrate this invention specifically. The detection of G6PD deficiencywas selected for this purpose because of the perceived need for areliable test for this purpose that can be conducted in the field,without instrumentation and in the absence of trained laboratorypersonnel. Assays for total serum cholesterol, beta lactomase activityand peroxidase activity are also exemplified.

SPECIFIC EXAMPLES Example 1

[0066] This test is performed on a lateral flow strip as pictured inFIG. 1A, having a ‘lyse”or, wicking, pad from which the sample flowsforward into the second, or substrate pad. The latter pad has tworegions. The first such region is a tightly confined substrate zone inwhich is movably pre-deposited all of the dried components that may beconventionally used in the art to enable G6PD in the sample to reducethe faint yellow dye nitroblue tetrazolium, to dark blue formazan. Therate of conversion of nitroblue tetrazolium to dark blue formazan is oneof several “wet chemistry” tests that has been used in the art tomeasure G6PD specific activity. The substrate pad also contains what isinitially a substrate-free zone, positioned at the farthest end of thestrip from the sample introduction point. As the sample picks upsubstrate and flows forward the initially substrate-free zone becomesthe “read” or endpoint zone when the sample containing reconstitutedsubstrate occupies it and forward flow ceases. Prior to the endpointzone, as the sample moves along the strip its forward flow momentumpicks up the dried substrate components and, as it moves to the end ofthe strip, reconstitution of the picked up dried ingredientsconcentrates in the fluid front, which rapidly becomes the endpoint or“read”zone where color develops. The area of the strip just prior to theread zone from which dried subtrate has been removed by the fluid frontthen becomes essentially substrate free, but meanwhile has been coloredred by the hemoglobin in the sample that passed through the zone.

[0067] A specific advantage of the lateral flow chromatography formatfor this test over a “wet chemistry” test method is realized because thesample of choice for G6PD activity determinations is blood. This isbecause, as earlier noted, blood cells contain the vast majority of eachhuman individual's G6PD. The blood cells must be lysed to make theenzyme which codes for G6PD available for the reaction. Lysis i.e.(splitting) of blood cells releases hemoglobin and imparts a red colorto the sample which must be removed or at the least, substantiallydiminished in intensity, when the test is conducted by wet chemistrymethods because the blue of formazan is extremely difficult, verging onimpossible, to discern visually in a diffuse liquid sample to which auniform red color has already been imparted. The blue color of formazanin such a diffuse liquid sample can readily be seen, as it emerges, as amere darkening of the initial red color; moreover, different individualsattempting to see the color change typically interpret any given testdifferently. In the chromatographic test herein described, however, thered color need not be removed because the reaction of sample andchromatographically reconstituted dried ingredients occurs in a welldefined flow region at the fluid front, so that when flow terminates atthe end of the chromatographic strip and excess fluid, if any, in thesample runs into a sink, two adjacent regions of the strip are clearlyvisible. The one closest to the end of the strip, the endpoint or “read”region, is a purplish blue color, while the adjacent region exhibits thered color of hemoglobin and the two are clearly discernable from oneanother.

[0068] In the actual tests performed with the G6PD test device, a humanwhole blood sample was drawn into a tube containing heparin to preventclotting of the sample. A portion of the sample was first assayed forG6PD activity using a clinical “wet chemistry” laboratory procedure ofthe prior art and, by ultraviolet spectrophotometric analysis wasconfirmed to have a normal human G6PD activity level of 116U/dl. Aportion of the sample was lysed by adding aqueous 10% Triton X-100 in avolume ratio of blood to lysing solution of 10:1. The resulting 10%Triton X-100, 90% whole blood lysate was incubated at 37° C. for 6 hoursto allow proteolytic degradation of its initial G6PD activity. Using thesame conventional “wet chemistry” laboratory procedure as used on aportion of the whole blood, this sample was found to have no G6PDactivity.

[0069] A second lysate sample was produced in the same manner as thefirst. Dilutions of the fresh lysate and the degraded lysate wereproduced with targeted G6PD activities of 104, 80, 40 and 20U/dl. Theactual activity of each of these samples was measured using the sameconventional “wet chemistry” lab procedure. The results of the freshlysate samples were consistent with the targets established for them,while the degraded samples consistently showed no activity.

[0070] To the sample receiving end of each of a series of separatelyprepared chromatographic G6PD strips mounted laterally on a supportthere was added 45 μl of each of the fresh and degraded lysate samples.The samples were allowed to flow chromatographically along the laterallyplaced strips to their terminal ends. As each sample reached theterminal end of a strip, timing was initiated and the time needed forthe visible purplish blue color to appear in the endpoint zone wasrecorded. The activity levels of the fresh lysate samples are shown inthe chart that is FIG. 2A. The times to purplish blue color appearanceare graphed against enzyme activity level for each fresh lysate samplein FIG. 2B. All concentration levels of the fresh lysate had G6PDactivity and eventually produced the purplish blue color in the endpointor “read” zone of the strip as expected.

[0071] These experiments were necessarily performed with pre-lysed bloodsamples because no G6PD deficient blood samples were available and itwas necessary to test a range of G6PD levels to validate the test. Lysisof blood samples at the sample introduction end of a chromatographicstrip is a procedure known in the art and is intended to be performed inthe known manner on these test strips in actual practice with samples ofunknown G6PD activity, thus obviating the need for any pre-test sampletreatment. The tests described above were considered necessary for thepurpose of establishing a timed endpoint so as to enable users of thetest strips in the field to distinguish readily between G6PD deficientblood and G6PD normal blood.

[0072] The clinically relevant G6PD deficiency level has heretofore beenfixed at 20 U/dl. The assay is designed to be run at ambienttemperatures as high as 37° C., which typically occur in warm climateswhere malaria infections are prevalent and the need to identifyG6PD-deficient individuals before prescribing malaria medication isacute. The rate of G6PD enzyme activity at 37° C. is known to be doublethat at normal room temperature of 25° C. For conducting the G6PDchromatographic test, a target activity level cutoff was set at 50 U/dlat room temperature (corresponding to 25 U/dl at 37° C.). By setting thetest endpoint at 70 seconds from the time the sample and entrainedreconstituted substrate reach the terminal end of the strip,discrimination between individuals with normal G6PD levels and thosewhose G6PD levels are clinically deficient can readily be made in thefield.

[0073] In the regions of the world where malaria is most prevalent, G6PDdeficiency is relatively common. The ease of performance of the test bynon-laboratory trained personnel and its speed combine to indicate thatthe use of the lateral flow chromatography G6PD test of this inventionwill have substantial value in ensuring that malaria-infectedG6PD-deficient individuals no longer receive medications for malariathat materially exacerbate their health problems.

Example 2

[0074] As previously noted, the measurement of cholesterol concentrationin a liquid sample can be performed by the enzyme-driven chromatographicassay of this invention in a manner similar to that for G6PD. A lateralflow test strip was constructed by depositing all of the reagentsconventionally used in wet chemistry tests for total serum cholesterolplus a color-producing ingredient required to produce a color change ata rate proportional to the concentration of cholesterol constituents.The phenol derivative used in this experiment was TOOS i.e.,3-(N-ethyl-3-methylanilino)-2-hydroxypropane sulfonic acid (Sigma,E-8631) which produces a blue/purple color that can be readilydistinguished above the hemoglobin background of a whole blood sample. Acommercially available panel of serum cholesterol standards (Sigma,C-0534) was run on these test strips to confirm that the time tovisualization was dependent on cholesterol concentration. A clear dosedependence was observed as seen in FIG. 3A and its plot, FIG. 3B.

[0075] To evaluate these test total cholesterol strips with a wholeblood sample, a panel of whole blood samples was constructed. Rabbitblood was used to simulate extreme hypocholesterolemia because therabbit native serum cholesterol level is very low, in this case 38mg/dl. For the balance of the tests at higher levels, this blood wascentrifuged and the serum fraction was withdrawn and separated intoaliquots. To each aliquot was added an equal volume of each of the serumcholesterol standards referred to above.

[0076] The resulting panel of adjusted whole blood samples was assayedwith a commercially available wet chemistry spectrophotometric assay(Thermo Trace, cholesterol concentration from 38 to 328 mg/dl). Each ofthese adjusted samples was lysed and assayed on a test strip asdescribed above. Again, a clear dose dependence was observed in thesewhole blood lysate samples. This dependent relationship is apparent fromFIG. 4A and the corresponding data plot, FIG. 4B.

[0077] In each of FIGS. 3B and 4B, y=cholesterol in mg. per dl. andx=time in seconds to visualization of color in the read zone. The symbolR² is the correlation coefficient of the data to the curve drawn.

Example 3

[0078] The beta-lactams are a class of antibiotics, includingpenicillins and cephalosporins, which contain a characteristicbeta-lactam ring structure. This structure interferes with enzymesneeded for the synthesis of peptidoglycan to produce defective cellwalls in dividing bacteria and renders these walls susceptible to lysisby osmotic pressure. One mechanism of bacterial resistance to theseantibiotics is the production of beta-lactamase enzymes, whichspecifically cleave the beta lactam ring, rendering the antibioticineffective and restoring the ability of the bacteria to multiplysuccessfully.

[0079] A lateral flow enzyme-driven chromatographic test strip to detectthe presence of β-lactamase was constructed by depositing all of thereagents required according to prescribed prior art wet chemistrymethodology, to produce a color change at a rate proportional to theconcentration of β-lactamase. As in Example 1, the liquid samplechromatographically reconstitutes the dry ingredients deposited on thechromatographic strip that measure beta lactamase activity, in thisexample a chromagnic cephalosporin (Oxoid, Sr0112C). Color formation isobserved in the reaction zone at the distal end of the strip only ifβ-lactamase was present in the original sample volume.

[0080] To confirm that the time to visualization of color with thesestrips was dependent on β-lactamase activity, a commercially availablepurified β-lactamase standard (Sigma, P-4524) was obtained,reconstituted, diluted to a range of β-lactamase activities and run onthese test strips. A clear dose dependence was observed as shown in FIG.5A and its data plot, FIG. 5B.

[0081] A sample of E. coli bacteria known to produce β-lactamase (ATCC#35218) was obtained and grown by culturing according to the directionsaccompanying it. The presence of β-lactamase activity in the culturedbacteria was confirmed by a conventional wet chemistryspectrophotometric assay. Next the concentrated culture was diluted, itscell concentration was calculated from a turbidity measurement and aseries of dilutions were run on the same test strips for beta-lactamaseactivity referred to earlier in this example. Here again, a clear dosedependence was observed in these diluted bacterial culture samples. Thisdependent relationship is apparent from FIG. 6A and its plot, FIG. 6B.In FIGS. 5B and 6B, y represents units of beta-lactamase activity and“Ln” is the natural logarithm of the number “x”, which represents themeasured time in seconds to visualization of color in the endpoint orread zone. In FIG. 6B, the symbol “E’ stands for a factor of 10, while“E” followed by a plus sign and a number signifies 10 to the positivepower of the number—e.g., 1.00 E+08 means 1×10⁸ “E” followed by a minussign and a number means 10 to the negative power of the number—thus inFIG. 6B where “R²=9.955E-01”; “E-01”=10⁻¹ and R² is 0.9955. In bothFIGS. 5B and 6B, R² is the correlation coefficient of the data to thecurve drawn.

[0082] Current methods of detecting beta lactamase enzymes require thatthe bacteria suspected of having developed antibiotic immunity becultured to expand the size of the bacterial colony and consequentlyexpand the amount of β-lactamase present. The colony is then incubatedwith disks that have been preimpregnated with chromogeniccephalosporins. Each disk absorbs a very small volume of the sample, andany β-lactamase enzyme present cleaves the chromogenic cephalosporin toproduce a pronounced color change in the disk.

[0083] In an effort to improve accuracy, efficiency and sensitivity ofthe method, superparamagnetic particles of sufficient magnetic moment tobe separated by exposure to the magnetic field of a rate earth magnetare conjugated to antibodies specific to the β-lactamase enzyme. Theseparticles are then added to a large volume of sample from an infectedpatient whose response to antibiotics is unsatisfactory. In the case ofresistant upper respiratory disease, for example, the sample is a nasalwash. When the suspect bacteria are urinary tract pathogens, a urinesample is ideal. The sample and the particles are incubated long enoughfor reaction to occur. The magnetic particles bearing theenzyme-antibody conjugates are separated by the magnetic field of therare earth magnet from unreacted particles and the liquid sample. Theseparticles are then released from the magnet and, resuspended in a smallvolume of buffer compatible with the test reagents on thechromatographic test strip for β-lactamase. Preliminary evaluations ofthe current practice with chromogenic cephalosporin-impregnated discsand enlarged bacterial colonies obtained by culturing the pathogens froma patient against the efficiencies expected to be realized by thetechnique described strongly suggest that markedly improved sensitivitywill be realized. Morever, obtaining the information rapidly may in manycases save a patient's life by indicating the need for a quick switch tomedication containing no β-lactam.

Example 4

[0084] Recent literature, including an article in the New EnglandJournal of Medicine (Prognostic Value of Myeloperoxidase in Patientswith Chest Pains: Vol. 349 #17, 1595-1604), highlights the potentialimportance of Myeloperoxidase as a marker of cardiac disease. As apotential diagnostic tool, a prototype enzyme-driven chromatographicassay was constructed to detect peroxidase activity in either serum orwhole blood. A lateral flow test strip was constructed by depositing allof the reagents required in existing wet chemistry methodology toproduce a color change at a rate proportional to the peroxidase activityof a test sample.

[0085] Briefly, glucose oxidase (Sigma, G-2133), 4-aminoantipyrridine(Sigma, 4382) and the phenol derivative TOOS (Sigma, E-8631) werecombined in solution, applied to the sample receiving end of achromatographic strip and dried. Similarly, a solution of glucose(Sigma, G-7528) was prepared, applied to the distal end of the samestrip in a volume such that an empty void space remained between thetreated regions and no mixing occurred, and dried. Upon reconstitutionof these dried components, as is known in the art, the glucose andglucose oxidase produce hydrogen peroxide which can be utilized by anyperoxidase activity in the sample to produce a characteristic purplecolor from the previously colorless phenol compound.

[0086] As hemoglobin, which may be present in small quantities in aserum fraction of whole blood and will be present in large quantities inunseparated whole blood has intrinsic pseudoperoxidase activity,additional reagents were included to insure this activity did notinterfere with the tests. Sodium nitrite is a strong oxidizing agentwhich will rapidly oxidize hemoglobin and eliminate its pseudoperoxidaseactivity as noted in U.S. Pat. No. 6,200,773. Sodium nitrate (Sigma,S-2252) was added to the first reagent zone of the above described teststrip in sufficient quantity to eliminate the pseudoperoxidase activityof a moderately hemolized human serum sample.

[0087] To determine whether the time to visualization with these stripswas dependent on the peroxidase activity in the test article, acommercially available horseradish peroxidase standard (Sigma, P-8375)was obtained and reconstituted. This standard was then diluted to arange of peroxidase activities in moderately hemolized human serum andrun on these test strips. A clear dose dependence in time tovisualization was observed as reflected in FIG. 7.

[0088] Using the dry chemistry, lateral flow-chromatography formatdescribed herein, most enzyme driven tests can readily be renderedeasier, faster and cheaper to perform. Combining chromatographicseparation techniques such as ion exchange, specific affinity, sizeexclusion and the like with the dry chemistry, lateral flow test formatwill in many instances produce even greater benefits.

[0089] Those skilled in the art of designing lateral flow chromatographytests will readily perceive ways of applying these techniques totransform much of what is now performed as clinical wet chemistry intolow cost, convenient tests performable in virtually any location byalmost anyone. In particular, various tests are well known to beperformable with alternative dry reagents to those specificallymentioned herein for use the substrate zone. Likewise, the ICT strip maybe prepared to enable the performance of additional steps prior to thecontact of sample with the dry ingredients in the substrate zone,without departing in any way from the spirit of this invention. It istherefore intended that the present invention be limited only by theappended claims.

We claim:
 1. A chromatographic strip adapted to the performance of a preselected enzyme-driven assay for a selected analyte, which strip is (a)adapted to be placed laterally in a supporting device, ensuring lateralflow along its length (b) constructed from a material that permitslateral chromatographic flow through its interstices, and (c) comprisesat least two pads sequentially positioned on an adhesive backing,namely, (1) a sample receiving pad adapted to receive a liquid samplewhich flows laterally and chromatographically therethrough and (2) asubstrate pad on which has been movably deposited within an area nearits interface with the preceding pad a dry mixture of components thathave heretofore been utilized in wet chemistry assays for the sameanalyte.
 2. A chromatographic strip according to claim 1 wherein thesample receiving pad is adapted to permit the addition of a lysing agentto a liquid blood sample introduced thereto, and to allow said lysedblood sample to flow chromatographically, in a lateral manner, into thesecond such pad.
 3. A chromatographic strip according to claim 1 whereina region of said first pad has been pretreated to remove from, or reducethe concentration in the liquid sample as it flows therethrough of asubstance known to interfere with, obscure the result of or otherwisehinder the performance of the intended assay if present therein.
 4. Achromatographic strip according to claim 1 wherein a region of saidfirst pad has been treated to concentrate the liquid sample as it flowsthere through by removing a portion of its liquid content.
 5. Achromatographic strip according to claim 1 wherein an intermediate padis interposed between the sample receiving pad and the pad and saidintermediate pad has been treated with an immovable deposit of at leastone substance that removes and binds at least one sample component thatwould otherwise interfere with or obscure the result of the assay.
 6. Achromatographic strip according to claim 2, the subtrate pad of whichcontains dry components that, when reconstructed by chromatographiclateral forward flow with a liquid sample of fresh human blood performan enzyme driven assay for G6PD activity on said sample.
 7. Achromatographic lateral flow assay for G6PD activity in human bloodcomprising the steps of a) applying to the sample receiving end of alaterally positioned chromatographic strip comprising at least twoabutting pads positioned on an adhesive strip, each constructed from amaterial that permits lateral chromatographic flow therethrough, aliquid sample of fresh human blood, b) applying to the sample a lysingagent to split open the red blood cells thereof, c) allowing the lysedblood sample to flow along said strip, into said the second pad, wherethe momentum of its forward flow picks up a movable predeposited drysubstrate mixture containing dry components heretofore utilized in wetchemistry clinical assays for G6PD activity, d) allowing the lysed bloodsample containing the dry substrate components in its forward flow frontto flow together along said strip to the terminal end thereof, and e)when the lysed blood sample and the dried substrate components reach theterminal end of said strip, noting the appearance of a distinct purplishblue color in the forward flow front due to the formation of formazan.8. An assay according to claim 6 wherein, in step (e) the time requiredfor development of purplish blue color after the sample andreconstituted substrate reach the end of the strip is measured and adetermination is made, based upon the measured time and the ambienttemperature during performance of the assay, of whether the person fromwhom the blood sample was obtained has normal or subnormal G6PDactivity, based on known time-temperature relationships to G6PD activitylevels.
 9. An assay according to claim 6 which is performed at ambienttemperature of approximately 37° C., wherein step (e) is permitted toproceed for 70 seconds from the time the sample and reconstitutedsubstrate reach the terminal end of the chromatographic strip and if nopurplish blue color has yet formed, the individual whose blood samplewas assayed is classified has having G6PD activity deficiency, but ifsuch color forms before the 70 second limit, the individual whose bloodsample was assayed is classified as having normal G6PD activity.
 10. Achromatographic lateral flow assay for a preselected analyte that isenzyme-driven and performed (a) by applying to the sample receiving endof a laterally positioned chromatographic strip comprising at least twopads positioned in abutting relationship on an adhesive backing, aliquid sample; (b) allowing said sample to flow through the first padand into the second pad, where the forward flow momentum of the samplepicks up and carries with it to the opposite end of said pad a mixtureof dry components which have been utilized in performing wet chemistryassays for the preselected analyte and (c) whereby the dry componentsare reconstituted by contact with the liquid sample and a colorindicative of the endpoint of the assay forms at the flow frontposition.
 11. A chromatographic lateral flow assay according to claim 2for measuring total serum cholesterol in mammalian blood, which striphas a substrate pad on which has been movably deposited within an areanear its interface with the preceding pad a dry mixture of componentsthat have heretofore been utilized in wet chemistry assays for totalserum cholesterol and a dye forming ingredient.
 12. A chromatographiclateral flow assay according to claim 1 for the measurement β-lactamaseactivity in bacteria cultures, which strip has a substrate pad on whichhas been movably deposited within an area near its interface with thepreceding pad a dry mixture of components heretofore utilized in wetchemistry assays for beta-lactamase activity and a dye-formingingredient.
 13. A chromatographic lateral flow assay according to claim2 for measuring peroxidase activity in human serum or whole blood, whichstrip has a substrate pad on which has been movably deposited within anarea near its interface with the preceding pad a dry mixture ofcomponents previously utilized in wet chemistry assays for peroxidaseactivity, plus a dye forming ingredient and sodium nitrite in sufficientquantity to eliminate pseudoperoxidase activity.
 14. A process forperforming a preselected enzyme-driven assay for a selected analytewherein the enzyme generally occurs in very small concentrations inmilieus where it is known to be present, which process comprises thesteps of (a) adding to a milieu in which the said enzyme is known tooccur sufficient aqueous-based liquid to provide a medium within whichmigration of said enzyme and/or particulate support material impregnatedwith or conjugated to, a ligand for said enzyme can take place, (b)adding to said medium an increment of particulate support materials of asize larger than any naturally occurring particles that may be presentin said medium, which particulate support materials have been conjugatedto or impregnated with a ligand for said enzymes, (c) allowing saidparticulate support materials which have been conjugated or impregnatedwith a ligand for said enzyme and said enzyme to incubate for a timesufficient to enable said enzyme to react with said ligand conjugated toor impregnated with said particulate support materials under conditionswhich are conducive to promoting contact between said enzyme and saidligand, (d) recovering from said medium said particulate supportmaterials upon which ligand-enzyme conjugates have formed, (e)dispersing said particulate support materials upon which ligand-enzymeconjugates have formed that were recovered in step (d) hereof in avolume of aqueous based medium of requisite sample size for conductingan assay, on a chromatographic strip adapted to the performance of anassay driven by the enzyme present in said ligand-enzyme conjugates, (f)applying said volume of aqueous based medium containing said particulatesupport material upon which ligand-enzyme conjugates have formed to thesample pad of a chromatographic strip according to claim 1 that isadapted to the performance of an assay driven by the enzyme present insaid ligand-enzyme conjugates, (g) allowing the assay to proceed and (h)reading its results using predetermined color standards
 15. A processaccording to claim 14 in which particulate support material is a porouspaper or plastic material and step (d) is performed by filtering outsaid particulate support materials upon which ligand-enzyme conjugateshave formed.
 16. A process according to claim 14 in which theparticulate support material is a porous paper or plastic material andstep (d) is performed by centrifuging said medium, separating out thelayer in which said particulate support materials upon whichligand-enzyme conjugates have formed and aspirating or filtering off anyliquid that is present in said layer.
 17. A process according to claim14 in which the particulate support material is a porous paper orplastic material and step (d) is performed by allowing said mediumcontaining said particulate support materials upon which ligand-enzymeconjugates have formed to rest and separate into layers by naturalsedimentation, followed by separating out the layer containing saidparticulate support materials upon which ligand-enzyme conjugates haveformed and removing any liquid present therein by aspiration orfiltration.
 18. A process according to claim 14 wherein the particulatesupport material is superparamagnetic particles and step (d) isperformed by exposing said medium to the action of a magnetic field ofsufficient strength to draw to it the particles on which ligand-enzymeconjugates have formed, the medium is removed by decantation oraspiration, and the isolated particles are released from the magnet andimmediately subjected to step (e).
 19. A process according to claim 18wherein the magnet is a rare earth magnet.